Project Summary In healthy individuals immune tolerance is maintained by populations of regulatory T-cells, including Foxp3+ regulatory T-cells (Treg). In individuals with relapsing-remitting multiple sclerosis (RR-MS), Treg numbers are normal in circulation but deficient at sites of autoimmune demyelination. Further, cells from these individuals function well ex vivo but fail to prevent autoimmunity in situ. These data implicate local defects in Treg homeo- stasis in the pathogenesis of RR-MS. However, the factors that regulate Treg homeostasis within peripheral tissues are unknown. A critical factor that governs Treg homeostasis is the cytokine interleukin 2 (IL-2) and downstream signaling through STAT5. Without this signaling, Treg revert to being conventional T cells (Tconv) or die. Since Treg cannot make IL-2 themselves, most of what they need comes from other T cells at sites of inflammation. How- ever, IL-2 has a very short half-life in vivo (<6 minutes) because it is very rapidly sequestered into the extracel- lular matrix (ECM) near where it is made. This is particularly a consequence of its binding to heparan sulfate (HS)-containing structures. Nonetheless, ambient IL-2 supports Treg stability and function at sites of recent inflammation, suggesting that these cells may be able to access this resource. We have identified a novel role for heparanase (HPSE), a cell-surface enzyme that binds and digests HS, in supporting Treg access to tissue IL-2. We find that HS-bound IL-2 is more potent than free (unbound) IL-2 in promoting Treg survival but only if cells express HPSE. Consistent with this, HPSE-/- mice have fewer Treg. Concomitantly, HPSE-/- Treg also have impaired phenotypic stability upon transfer in vivo. These effects of HPSE on Treg homeostasis may be mediated at the level of interactions between IL-2 and the IL-2 receptor (IL-2R). We find that HS-bound IL-2 potently induces STAT5 signaling in wildtype, but not HPSE-/- Treg. Because IL-2/IL-2R interactions typically trigger internalization and clearance of IL-2 as well as signal- ing, our preliminary data suggest that HPSE may stabilize HS/IL-2/IL-2R complexes in ways that prolong IL-2 signaling. This may be a fundamental mechanism governing Treg survival and function in peripheral tissues. In light of these exciting preliminary data, we hypothesize that FoxP3+ Treg use HPSE to access IL-2 bound to HS within tissues. To test this hypothesis, in Aim 1 we will define the role of HPSE in Foxp3+ Treg homeosta- sis and function. Then, in Aim 2 we will elucidate how HPSE potentiates IL-2R signaling. Together, these aims represent a bold, imaginative, and unconventional approach to studying immune homeo- stasis in peripheral tissues. This work will provide the mechanistic foundation needed to support R01 level re- search into the role of HPSE in human MS and the development of novel therapies targeting HPSE to promote immune tolerance.